The functional role of CST1 and CCL26 in asthma development

Abstract Background Asthma is the most common chronic disease in children with an increasing prevalence. Its development is caused by genetic and environmental factors and allergic sensitization is a known trigger. Dog allergens affect up to 30% of all children and dog dander‐sensitized children show increased expression of cystatin‐1 (CST1) and eotaxin‐3 (CCL26) in nasal epithelium. The aim of our study was to investigate the functional mechanism of CST1 and CCL26 in the alveolar basal epithelial cell line A549. Methods A549 cells were transfected with individual overexpression vectors for CST1 and CCL26 and RNA sequencing was performed to examine the transcriptomics. edgeR was used to identify differentially expressed genes (= DEG, |log2FC | ≥ 2, FDR < 0.01). The protein expression levels of A549 cells overexpressing CST1 and CCL26 were analyzed using the Target 96 inflammation panel from OLINK (antibody‐mediated proximity extension–based assay; OLINK Proteomics). Differentially expressed proteins were considered with a |log2FC| ≥ 1, p < .05. Results The overexpression of CST1 resulted in a total of 27 DEG (1 upregulated and 26 downregulated) and the overexpression of CCL26 in a total of 137 DEG (0 upregulated and 137 downregulated). The gene ontology enrichment analysis showed a significant downregulation of type I and III interferon signaling pathway genes as well as interferon‐stimulated genes. At the protein level, overexpression of CST1 induced a significantly increased expression of CCL3, whereas CCL26 overexpression led to increased expression of HGF, and a decrease of CXCL11, CCL20, CCL3 and CXCL10. Conclusion Our results indicate that an overexpression of CST1 and CCL26 cause a downregulation of interferon related genes and inflammatory proteins. It might cause a higher disease susceptibility, mainly for allergic asthma, as CCL26 is an agonist for CCR‐3‐carrying cells, such as eosinophils and Th2 lymphocytes, mostly active in allergic asthma.


| INTRODUCTION
Asthma is among the most common chronic diseases worldwide in children and adults.Onset is most often in childhood, and its prevalence has increased in recent years.The disease is characterized by chronic inflammation of the lower airways, variable airflow obstruction and airway hyperresponsiveness, leading to symptoms such as wheezing, shortness of breath and coughing.Wheezing episodes are mostly caused by rhinovirus infections that may result in a broken epithelial barrier and lead hereby more easily to inflammations. 1,2Asthma can be divided into subgroups according to the type of inflammation driving the disease.Childhood asthma is divided into allergic asthma (Th2 inflammation), nonallergic and mixed, with allergic asthma being the most prevalent type of asthma in children. 3 The disease leads to life-long consequences for patients, including loss of time for work and education, impaired quality of life, and high costs for society. 4The most common asthma treatments nowadays are bronchodilators and inhaled corticosteroids, which act as airway inflammation suppressors due to the downregulation of pro-inflammatory cytokines and inflammatory genes. 5The development of asthma stems from genetic and environmental factors.Though no exact genetic mechanisms are currently known, several genes have been suggested to be asthma susceptibility genes. 6llergic sensitization is a known trigger for asthma development.One major cause are dog allergens which affect up to 30% of all children and adolescents. 7A Swedish birth cohort study including children between 4 and 16 years of age showed that allergic sensitization to dog is increasing from 4.8% to 22.6%. 8Another Swedish population-based cohort study including children between 11 and 12 years of age showed a sensitization to dog allergens of 31.5%. 9n a recent study, we investigated nasal epithelium of 54 dog dander-sensitized children and 20 healthy controls aged 10-18 years. 10Transcriptome analysis identified 321 genes that were significantly differentially expressed (DEG) between the case and control groups of those were 108 genes significantly upregulated and 213 significantly downregulated.Among the 321 DEG, cystatin-1 (CST1, Cystatin-SN) and chemokine (C-C motif) ligand 26 (CCL26, Eotaxin-3) showed the highest upregulation.
CST1 can be found in biological fluids and leads to reversible and competitive inhibition of cysteine proteinases known as cathepsins. 11Furthermore, cystatins have potent immunomodulatory functions by inducing the synthesis of TNF-α and IL-10. 124][15] A recent study by Wang et al. 16 identified CST1 as a potential biomarker for asthma by analyzing 6 datasets of asthmatic patients.Upregulation of CST1 in bronchial or nasal epithelium of asthmatic patients, highlights the role of CST1 in the pathophysiology of asthma. 17,18CL26 is a selective agonist for CC chemokine receptor 3 (CCR3) and attracts CCR-3-carring cells like eosinophils, 19 basophils 20 and Th2 lymphocytes, 21 the most prominent cell types in allergic asthma. 224][25] Even though several studies showed an association between increased levels of CST1 and CCL26 and the development of asthma, it was to our knowledge so far not possible to understand the contribution of CST1 and CCL26 in the disease development.Obtaining more knowledge about the mechanistic insights of those genes, it might be possible to identify whether CST1 and CCL26 can be potential drug targets for asthma treatment.Therefore, the aim was to investigate the functional mechanisms of CST1 and CCL26 in an alveolar basal epithelial cell line to understand their function in asthma pathogenesis better.

| Plasmids and transfections
Expression plasmids containing the human CST1 and CCL26 as well as the empty vector pCMV6-entry as control were obtained from OriGene Technologies Inc. (Table 1).A549 cells were transfected with 1 µg plasmid DNA containing CST1 (CST1 overexpression) or CCL26 (CCL26 overexpression) separately in 24-well plates using Lipofectamine® 3000 (Thermofisher Scientific, USA), according to the manufacturer's instruction.The cells were harvested for subsequent experiments 48 h following transfection.All experiments were performed in biological quadruplicates.

| Reverse transcription and realtime PCR (rt-qPCR)
Total RNA was extracted with the Qiagen RNeasy Micro kit (Hilden, Germany) 48 h after transfection and the concentrations were measured using Qubit TM Flex Fluorometer (Thermofisher Scientific).RIN values were obtained from Qsep100 Bio-Fragment Analyzers (Bioptic, Inc.) and ranged between 8.9 and 10.Overexpression was confirmed using rt-qPCR (Figure S1).cDNA was synthesized using SuperScript III First-Strand Synthesis Super-Mix (Thermofisher Scientific), according to the manufacturer's protocol.For rt-qPCR, 1 µL of cDNA was used in a 20 µL reaction volume, TaqMan gene expression Master Mix and pre-developed TaqMan gene expression assays (Thermofisher Scientific).All samples were run in triplicates according to the manufacturer's instructions.Each reaction was normalized to the GAPDH and PPIA content separately and the ΔΔCT method 26 was used to determine the fold induction over cells transfected with the empty vector (control samples).

| Transcriptome library preparation and sequencing
In total, 12 samples were included (4 biological replicates of CST1, 4 biological replicates of CCL26, 4 biological replicates of pCMV6-Entry).Library preparation and sequencing were performed by the National Genomics Infrastructure (NGI) in Stockholm, Sweden.The libraries were prepared with the Illumina® TruSeq Stranded mRNA library preparation kit with 330 ng total RNA of each sample by Agilent Bravo workstation, as based on the NGI sample recommendations.The average library length for CST1 was equal to 362 bp, for CCL26 equal to 400 bp and for pCMV6-entry equal to 395 bp.Sequencing was performed on an Illumina Ⓡ NovaSeq.6000 platform using the NovaSeq.6000 S4 Reagent Kit (35 cycles) (Illumina).

| Proteomic analysis
The protein expression of A549 cells overexpressing CST1 and CCL26 separately as well as the empty vector was analyzed with the antibody-mediated proximity extension-based assay OLINK (Proteomics, Uppsala, Sweden) using the Target 96 inflammation panel.RIPA lysis buffer (Merck) supplemented with proteases inhibitors (Roche) was used to collect the cell lysate 48 h after transfection.Lysis buffer only was included as negative control.The measurements were conducted in duplicates at the affinity proteomics-Stockholm facility as previously described. 38A B L E 1 Plasmids obtained from OriGene.The protein expression levels are presented as normalized protein expression (NPX) values in a log 2 -scale.If 15% of the samples from one protein had values below the limit of detection (LOD), the proteins were excluded from the analysis (Table S3).

Catalog number
The difference between the protein expression levels of cells overexpressing the gene of interest or the control vector, delta-NPX, were compared using twotailed t-test in Microsoft Excel.Delta-NPX is presented as log 2 FC and proteins with |log 2 FC | ≥ 1 and p < .05were considered as differentially expressed proteins.ClustVis (v.1.0) 39was used to generate the heatmaps of the expression data.

| RESULTS
A549 cells were transfected with either CST1 or CCL26 to investigate the functional mechanism of these genes (Figure 1).
F I G U R E 1 Flow chart of the study.A549 cells were separately transfected with the expression plasmids containing human CST1 and CCL26 as well as the empty vector pCMV6-entry as control, obtained from Origene Technologies Inc.In total, 12 samples were included (4 biological replicates of CST1, 4 biological replicates of CCL26, 4 biological replicates of pCMV6-Entry).On the one hand, RNA was extracted with the Qiagen RNeasy Micro kit 48 h after transfection and on the other hand, the cell lysates were collected with RIPA lysis buffer.Transcriptomic analysis was conducted via RNA sequencing and proteomic analysis via the Target 96 inflammation panel from OLINK.The transcriptomic analysis was conducted in biological quadruplicates and the proteomic analysis in biological duplicates.The results identified a downregulation of type I and III interferon genes as well as differentially expression of inflammatory proteins.Created with BioRender.
The overexpression was confirmed with rt-qPCR (Figure S1) and downstream effects were evaluated with RNAseq.edgeR analysis was performed on the RNAseq data to obtain the differentially expressed gene profiles ( | log 2 FC | ≥ 2, FDR < 0.01).The overexpression of CST1 in A549 cells resulted in a total of 27 differentially expressed protein coding genes.Out of the 27 DEG, 1 was upregulated and 26 downregulated (Figure 2, Table 2, Figure S2A, Table S1).An overexpression of CCL26 resulted in a total of 137 differentially expressed protein coding genes of those no gene was upregulated and 137 were downregulated (Figure 2, Table 2, Figure S2B, Table S2).
Gene ontology enrichment analysis was performed for all downregulated genes (log 2 FC ≤ −2, FDR < 0.01) due to the overexpression of CST1 and CCL26, separately, based on biological processes.
For the protein coding downregulated genes following CST1 overexpression (n = 26), defense response to virus, defense response to symbiont, negative regulation of immune effector process were the top three biological processes (Figure 3A).The downregulated protein coding genes due to CCL26 overexpression (n = 137) showed as top 3 biological processes response to virus, defense response to virus and defense response to symbiont (Figure 3B).The RNAseq results were confirmed via rt-qPCR and showed a downregulation of IFNB1 as well as IFNL1-3 for the samples overexpressing CST1 (Figure 4) as well as CCL26 (Figure 5).The network analyses of the downregulated genes for both CST1 and CCL26 overexpression models are shown in Figure 3C,D.
All downregulated genes after overexpression of CST1 overlap with the downregulated genes after CCL26 overexpression (Figure 6).
Protein levels of the A549 cells overexpressing CST1 or CCL26 were analyzed with the antibody-mediated proximity extension-based assay OLINK using the Target 96 inflammation panel.In A549 cells overexpressing CST1, only CCL3 was differentially higher expressed with a log 2 FC of 1.99, and no protein was differentially lower expressed.Among the samples overexpressing CCL26, in total 5 proteins were differentially expressed, HGF was higher expressed with a log 2 FC of 1.10 and CXCL11, CCL20, CCL3 and CXCL10 were lower expressed with a log 2 FC of −1.73, −1.64, −1.12, and −1.07, respectively (p < .05,Table 3, Tables S4, S5, Figure S3).

| DISCUSSION
Increased expression of CST1 and CCL26 has been previously shown in nasal epithelium of children with asthma and allergy, 10 however, the functional mechanism of how it contributes to asthma development is so far not well understood.Therefore, our study examined the transcriptomics and inflammatory proteins due to the overexpression of CST1 and CCL26 separately in the alveolar basal epithelial cell line A549.Significant F I G U R E 2 Volcano plot of the differentially expressed genes after the overexpression of (A) CST1 and (B) CCL26, separately.CST1 and CCL26 are in the upper right corner of the respective volcano plot.Gray: nonsignificantly expressed genes, blue: significantly downregulated genes, red: significantly upregulated genes with a |log 2 FC | ≥ 2. FDR, false discovery rate.downregulation of genes involved in type I and III interferon signaling pathway and several interferonstimulated genes, especially due to the overexpression of CCL26 was seen.Also on protein level, the overexpression of CCL26 induced slightly more differentially expressed proteins.Due to the downregulation of interferon type I and III genes, we hypothesize that the overexpression of CST1 and CCL26 results in a more severe airway inflammation and disease progression.
In our study, the transcriptomic profile after overexpression of CST1 induced an upregulation of one (CST2) and a downregulation of 26 protein coding genes.CST2 (Cystatin-SA), a thiol protease inhibitor, is the significantly upregulated gene and, like CST1 protects against allergens, viral and bacterial proteases and is highly expressed in salivary glands. 40,41Both CST1 and CST2 consist of 141 amino acids and include cysteine protease inhibitor, proteinase inhibitor I25A and I25B, type 2 and phytocystatins and CST1 also a Myb DNAbinding domain 42,43 and it could be previously seen that the genes are closely linked to each other. 44A recent publication from Nocera et al. 45 identified overexpression of CST2 in patients with chronic rhinosinusitis with nasal polyp (CRSwNP) compared to controls.
The overexpression of CCL26 in the alveolar basal epithelial cell line A549 caused no upregulation of any gene.
An enrichment of genes involved in interferon signaling, including interferon-stimulated genes was seen to be downregulated in the alveolar basal epithelial cells after overexpression of CST1.It resulted among others in the downregulation of the type I and III interferons IFNB1 and IFNL1 (IL-29) and IFNL2 (IL-28A).The function of interferons is generally the induction of antiviral enzymes and interferonstimulated genes. 46Several interferon-stimulated genes linked to viral replication were downregulated in the CST1 overexpressing cells.Among those, IFITM1 (interferon-induced transmembrane protein 1) that inhibits the viral entry via the endocytic vesicle membrane which fuses with incoming viruses. 47Moreover, IFIT2, inhibits viral protein production during viral mRNA translation. 48The viral replication of different RNA and DNA viruses is suppressed by RSAD2 (viperin) a radical SAM (S-adenosyl-L-methionine) enzyme, due to its interaction with cellular and viral proteins. 49OAS2 (oligoadenylate synthetases) degrades viral genomes 50 and BST2 (tetherin), a transmembrane protein, prevents  at the last stage of the viral cycle, the release of virions. 51urthermore, the HLA class I (major histocompatibility complex, class I, human leukocyte antigen, MHC class I) genes HLA-B and -F were downregulated after the overexpression of CST1.MHC class I genes present antigens to cytotoxic CD8 + T cells, which activates them to eliminate infectious cells and to produce inflammatory cytokines like IFNγ. 52In contrast to our results, previous findings showed increased HLA-F surface expression in severe asthmatic patients. 53However, a downregulation of MHC class I could be observed in human blood cell lines as well as the alveolar basal epithelial cell line A549 after the infection with influenza A and B, 54 nevertheless, our cells overexpressed CST1 and had no virus infection.We can speculate that an overexpression of CST1 mimics a virus infection and causes therefore a downregulation of HLA-F.
It is known that rhinovirus especially causes wheezing symptoms in preschool children and infection can increase the risk for asthma. 55Our findings of the downregulation of genes involved in viral defense mechanism following CST1 overexpression, suggest that higher levels of CST1 may facilitate viral entry.The consequence may be an increased rhinovirus load that might lead to the development of asthma later in life.As a rhinovirus infection cause an inflammation of the upper and lower airways, it is a potential trigger in the development of asthma. 56enes downregulated due to CST1 or CCL26 overexpression overlap (Figure 6).Comparing the significantly downregulated genes after CCL26 and CST1 overexpression, almost all of these 137 genes were significantly downregulated also due to CST1 overexpression, however with a log 2 FC below our cut-off (data not shown).That might indicate that CST1 as well as CCL26 have similar effects or are regulated by each other.As CCL26 attracts eosinophils, this interaction is supported by previous studies showing that CST1 progresses the migration of eosinophils and might play a role in the development of chronic rhinosinusitis. 57,58n addition to the downregulated genes already discussed, the interferon type III (IFNL3 (IL-28B)) gene as well as 110 more genes, were downregulated due to CCL26 overexpression.Genes with a function within interferon signaling or similar are discussed in the following.RIG-I (DDX58), a cytosolic sensor of viral RNA acts via the adapters MAVS and STING and activates the type I interferon pathway, also via IRF7. 59he interferon-repressed genes STAT1 and STAT2 are transcription factors in the interferon signaling pathways.Via the JAK/STAT pathway, the transcription factor complex gets activated, which consists of SH2phosphotyrosine-mediated heterodimer STAT1 and STAT2 as well as IRF9. 60USP18 (Ubiquitin-specific protease) regulates negatively the IFN induction 61 and ISG20, a 3′−5' exonuclease, degrades viral genes and genome. 62Furthermore, several studies showed that MX1 suppresses influenza A virus infections, 63,64 MX2 HIV-1 infections 65,66 and IFI6 the replication of flavivirus. 67IFITM3 inhibits the viral entry like previously mentioned IFITM1 and IFI16 is a sensor for viral DNA and RNA. 47,68The poly-ADP ribose polymerase (PARP) suppresses viral replication by building complexes with the ribosome. 69The genes downregulated due to CCL26 overexpression are again linked to viral entry as well as inflammation.An increase viral load due to an enlarged viral entry is known to increase the risk for asthma development later in life. 55Inflammation pathways like JAK/STAT, involved in the regulation of Th2 cells 70 are moreover a potential target for the treatment of allergic diseases. 71ur results indicate that the overexpression of CCL26 as well as CST1 causes a downregulation of several antiviral response genes which has previously been seen in patients with asthma and allergy.Hwang et al. 72 investigated sinonasal epithelial cells of patients with chronic rhinosinusitis with and without nasal polyps comparing it to healthy controls.A decreased expression of IFNB, IFNL1, IFNL2, OAS, MX1 and RSAD2 was seen in both RNA and protein levels in sinonasal mucosa with inflammation compared to normal mucosal tissue.Although the findings are from nasal epithelium, they are in line with our hypothesis that the overexpression of CCL26 causes a more severe asthma phenotype, based on previous findings in nasal epithelium. 10Furthermore, a reduced induction of IFNL genes could also be seen in asthmatic primary bronchial cells and alveolar macrophages after rhinovirus infection 73 as well as for interferon beta. 74ue to the findings of this current and previous studies, we hypothesize that the overexpression of CST1 and CCL26, lead to a downregulation of interferon type I and III genes, which in turn may cause a more severe airway inflammation and progression of the disease.Our findings are in line with the theory proposed by Holt et al., 75 showing that lower interferon levels at birth caused a more severe infection of the lower respiratory tract later in life.That might repose that the viral infections cannot be well controlled as genes involved in the inhibition of the viral replication cycle are not sufficiently expressed, and it results therefore in a deficient immune response and a higher disease susceptibility.
We hypothesize that heavily increased expression of CCL26 causes a more severe phenotype of asthma, especially allergic asthma, as CCL26 is also an agonist for CCR-3-carrying cells, like eosinophils 19 and Th2 lymphocytes, 21 the most prominent cell type in allergic asthma.Asthma is often paired with allergic sensitization 76 and the most prevalent type asthma in children. 3 Due to the damage of the airway mucosa upon viral infection, patients are more susceptible to allergens. 77ncreased expression of type I interferon could also be seen in nasal epithelium of children compared to adults infected with SARS-CoV-2, 78 which indicates different mechanism between adults and children.However, an increase of IFNL in a cohort of wheezing children aged 5-18 years was associated with asthma exacerbation. 79evertheless, studies in mice models 80 and human peripheral blood mononuclear cells 81,82 showed that an administration of type I and III interferons decreased inflammation in the airways due to a decreased IL-13 secretion and inhibition of Th2 cells.These data indicate that the role of interferon levels in asthma development has yet to be fully elucidated.Different findings regarding an up-or downregulation of type I and III interferons in association with asthma have been identified in several studies (Table 4).Due to the fact that several genes have been associated as asthma susceptibility genes, 6 it might be speculated that increased or decreased levels of type I and III interferon genes as well as interferon-stimulated genes are linked to the enormous overexpression of a specific gene, in our study CST1 and CCL26.Therefore, further studies investigating asthma associated genes and their downstream effects are warranted.It would be hereby possible to identify the function of more asthma susceptibility genes.
On the protein level, the OLINK analysis showed that the overexpression of CCL26 induced more differentially expressed proteins than CST1, which is consistent with the transcriptomic data, strengthening our hypothesis that especially CCL26, causes a more severe airway inflammation, particularly in allergic asthma.
Due to the overexpression of CCL26, the chemokines CXCL10, CXCL11, CCL20 and CCL3 were significantly downregulated, supporting hereby the findings of downregulation for CXCL10, CXCL11 and CCL20 on RNAlevel (log 2 FC < −1.6).A previous study of cord blood found associations between higher CXCL10 levels in infancy and asthma later in life, contradictory to our findings, but decreased levels of CXCL11 that were associated with allergic sensitization later in life, in line with our study. 85In plasma extracellular vesicles of allergic patients, CCL20 was among the downregulated biomarkers 86 what is in line with our results.Nevertheless, higher CCL3 concentration in serum could be observed in patients with allergic rhinitis, 87 only seen for the samples overexpressing CST1.
Though, the previous findings were observed in cord blood and serum, to the best of our knowledge, in airway tissue the information on their expression levels is limited.
A previous study in A549 cells investigated the overexpression of the Toll-like receptor (TLR), that has an important role in the innate immune system, and observed a decreased expression of among others CXCL10 and CCL20 in the cell supernatant due to the overexpression of TLR10, 88 similar to our findings in cell lysate.Furthermore, a meta-analysis of nasal epithelium from children between 6 and 16 years of age, demonstrated decreased expression of CXCL11 and CXCL10 in patients with obesity-related asthma 89 and is also in line with our hypothesis that the overexpression of CCL26 causes a more severe asthma phenotype, based on previous findings in nasal epithelium. 10n a study of A549 cells infected with respiratory syncytial virus (RSV), higher concentrations of CXCL10 and CCL3 were identified in the cell supernatant 24 h after RSV infection. 90Our study observed a downregulation of CXCL10 and CCL3 due to the overexpression of CCL26, but the overexpression of CST1 induced a significant upregulation of CCL3.The cells in the current study were only overexpressed with CST1 and CCL26 separately, but not infected with any virus and intracellular proteins were measured.
T A B L E 4 Overview about findings regarding increased and decreased levels of type I and III interferons.

Downregulation of type I and III interferons Study population Results
Hwang HGF with its function to decrease airway inflammation and airway remodeling, 91 was increased expressed in A549 cells overexpressing CCL26, and is against our hypothesis that an overexpression CCL26 causes a more severe asthma phenotype, nevertheless it was slightly lower expressed due to the overexpression of CST1, but not significant.In the current study, four proteins (CXCL11, CCL20, CCL3, CXCL10) that were downregulated due to the overexpression of CCL26, are slightly upregulated due to the overexpression of CST1, however, only significantly different for CCL3.Overall, the findings pointing to the direction that the overexpression of both CST1 as well as to a stronger extend CCL26 cause an alteration of genes involved in type I and III interferon response as well as inflammatory proteins.Also seen on mRNA level as all downregulated genes due to the overexpression of CST1 overlap with the downregulated genes after CCL26 overexpression.

| Clinical implications and future perspectives
Overall, our findings increase the knowledge of the underlying mechanism of the overexpression of CST1 and CCL26, namely, decreasing the expression levels of interferons, which are important components of the viral defense.Wheeze is a common symptom in children, most commonly induced due to a rhinovirus infection and associated with asthma development. 56Our results may indicate that the overexpression of CST1 and CCL26 causes a more severe airway inflammation, particularly in allergic asthma, as well as an impaired immune system.Therefore, CST1 and CCL26 might be considered as potential drug targets for asthma treatment.A direct downregulation of CST1 and CCL26, could possibly increase the interferon levels, what should be investigated in future drug discovery studies.Targeted treatment can mitigate the progression of the disease as well as delay onset.This opens for the possibility to design treatments that target the cause of asthma and not just the symptoms.

| Strength and limitations
3][94] However, the cells are obtained from a donor with lung adenocarcinoma, therefore, the cells differ from healthy primary cells.Nevertheless, the differentially expressed genes of both overexpression experiments, did not show an enrichment of pathways related to cancer.Furthermore, a previous study from Roberts et al., 95 showed that nasal and bronchial epithelial cells response similar to rhinovirus infections.The gene expression profiling of nasal and bronchial brushings from patients with airway disease and healthy controls, also showed an overlap of 98.2% DEG between the matched samples. 96Both studies show that the alveolar basal epithelial cell line A549, used in our study, represents a meaningful model to investigate previous findings from the nasal epithelium.
A downregulation of the type I and III interferons due to mycoplasma contamination can be excluded, as the cells were regularly tested for contamination.
We measured the protein expression levels of cells overexpressing CST1 and CCL26 separately via the Target 96 inflammation panel from OLINK to explore the findings on protein level, what is a strength of our study.OLINK is a highly sensitive method that allows the measurement of several proteins at the same time.In total, only 45/92 could be included in the analysis as the rest of the proteins did not pass the LOD, though this is to be expected when doing measurements from cell lysates.Nevertheless, our OLINK analyses had a small sample size with two samples in each group, which is a limitation of the study.For the transfection of A549 cells, we used the transfection delivery vehicle as transfection control to avoid experimental biases, strengthening our experimental approach.However, our study might include a confirmation bias as we have four biological replicates for the RNA sequencing samples, however, no technical replicates.Furthermore, it can be possible that we have a replicate bias for the RNA sequencing and OLINK samples as the experiments were performed in the same manner, however, not on the same day.

| CONCLUSION
Our study investigated the functional effects of downstream targets following the overexpression of CST1 and CCL26 separately in the alveolar basal epithelial cell line A549.We found a downregulation of type I and III interferons and several interferon-stimulated genes.It indicates that patients with upregulated CST1 and especially CCL26 levels also confirmed on protein level, have a deficient immune response and a higher disease susceptibility.A specific downregulation of those genes might present a drug target to increase in return the immune response.

F I G U R E 3
Gene ontology enrichment analysis for the downregulated genes due to (A) CST1 overexpression (n downregulated = 26) and (B) CCL26 overexpression (n downregulated = 137).The x-axis represents the GeneRatio, number of gene count indicated by the dot size, adjusted p-value indicated by color (red = high significant, blue = low significant); the y-axis represents the gene ontology biological processes.Network analysis of the relationship between the enriched pathways for the downregulated genes due to (C) CST1 overexpression (n downregulated = 26) and (D) CCL26 overexpression (n downregulated = 137).Two nodes are connected if they share 20% (default) or more genes.Darker nodes = more significantly enriched gene sets, bigger nodes = larger gene sets, thicker edges = more overlapped genes.F I G U R E 4 Confirmation of the RNAseq results via rt-qPCR for the CST1 samples.F I G U R E 5 Confirmation of the RNAseq results via rt-qPCR for the CCL26 samples.

F I G U R E 6
Venn diagram of differentially expressed downregulated protein coding genes after either CST1 or CCL26 overexpression.
Differentially expressed genes after overexpression of CST1 and CCL26, separately.
T A B L E 2